The PIV3 F glycoprotein expressed by a recombinant baculovirus is antigenically authentic as determined using a panel of PIV3 F specific monoclonal antibodies. Only a low level of antibody was induced by immunization of animals with insect cells infected with the baculovirus F recombinant and the animals were moderately protected against PIV3 challenge. Although the baculovirus expression system appears to be a reasonable source of authentic PIV3 F protein, its poor immunogenicity suggests that its use as a subunit vaccine is problematic. The complete nucleotide sequence of the JS strain of PIV3 was determined and compared to that of the prototype A/Wash/1957 PIV3 strain. Regulatory regions of the genomes were highly conserved, the 3' and 5' non-coding regions showed up to 14% sequence divergence, and the coding regions of all genes other than P showed only 1 to 2% amino acid divergence. A complete cDNA copy of the JS PIV3 wild type is being assembled. The attenuated cold passage 12 mutant of the JS strain of PIV3 was also sequenced and thirteen nucleotide changes that occurred during passage in vitro were identified. The cp12 mutant sustained three nucleotide changes in regulatory regions of the genome and 7 changes in the coding region that lead to amino acid substitution. The nucleotide or amino acid changes responsible for the temperature-sensitive (ts) and attenuation (att) phenotypes remain to be determined. A technique for sequence analysis of a hypervariable region of the 5' non-coding region of the F gene has been developed that can identify PIV3 isolates from vaccinees as vaccine-derived or community-derived virus. This will be especially helpful in identifying the origin of isolates that have lost one or more phenotypes of a candidate vaccine virus (such as the ts property) following replication in vaccinees.